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Nitric Oxide Prevents a Pathogen-Permissive Granulocytic Inflammation During Tuberculosis

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Nitric Oxide Prevents a Pathogen-Permissive Granulocytic Inflammation During Tuberculosis ARTICLES PUBLISHED: 15 MAY 2017 | VOLUME: 2 | ARTICLE NUMBER: 17072 Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis Bibhuti B. Mishra1, Rustin R. Lovewell1,AndrewJ.Olive1, Guoliang Zhang2, Wenfei Wang2, Eliseo Eugenin3, Clare M. Smith1,JiaYaoPhuah1, Jarukit E. Long1, Michelle L. Dubuke4, Samantha G. Palace1, Jon D. Goguen1,RichardE.Baker1, Subhalaxmi Nambi1, Rabinarayan Mishra5, Matthew G. Booty1,ChristinaE.Baer1, Scott A. Shaffer4, Veronique Dartois3,BethA.McCormick1, Xinchun Chen2,6* and Christopher M. Sassetti1* Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen’s environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion. espite the ability of Mycobacterium tuberculosis (Mtb)to The primary protective role of NO is anti-inflammatory sustain a persistent infection that can last for decades, most Upon infection with Mtb, mice deficient for inducible NO Dindividuals remain asymptomatic because their immune generation—either due to genetic deletion of the Nos2 gene6 or response effectively contains the pathogen. Only a fraction of chemical inhibition with aminoguanidine (AG)7—suffer from those infected with Mtb will develop the progressive inflammatory high bacterial burdens, weight loss and progressive granulocyte disease, tuberculosis (TB). In these individuals, continual bacterial accumulation that correlates with increased IL-1α and IL-1β replication and progressive necrosis produce cavitary lesions (Supplementary Fig. 1a–e). The granulocytes that accumulate − − contiguous with the airway, which allow bacteria to exit the host in the lungs and spleens of Nos2 / animals were predominantly and infect others1. All infected individuals mount a robust CD11b+, Gr1hi, Ly-6Chi and Ly-6Ghi and had the nuclear immune response to the pathogen, but the immune mechanisms morphology of neutrophils (Fig. 1a and Supplementary Fig. 1f–i). that differentiate protection from disease remain unclear. A smaller proportion of Gr1int myeloid cells with more hetero- Protective immunity to TB requires T cell-derived interferon geneous cytological appearances were also present, as shown in gamma (IFN-γ), which induces the expression of nitric oxide other susceptible mice8–10. Bone marrow chimaeric mice were synthase 2 (Nos2), required for generating nitric oxide (NO) in created to identify the source of protective NO and determine if macrophages. Animals lacking either of these factors suffer this compound’s cell-intrinsic antimicrobial activity is responsible from severe TB disease characterized by high bacterial loads for inhibiting disease. Haematopoietic reconstitution of irradiated − − and granulocytic inflammation2. This correlation between neutro- Nos2 / mice with wild-type, but not Nos2-deficient, bone phils, Mtb burden and pathology is a common feature of both marrow cells inhibited all metrics of disease (weight loss, neutrophil animal models and human TB (refs 3–5). Because NO can kill influx and bacterial replication; Fig. 1b and Supplementary Fig. 2a). Mtb in axenic culture, most models of protective immunity posit To determine if the loss of Nos2 diminished the cell-autonomous that this mediator primarily acts by inhibiting bacterial replication, antimicrobial capacity of the macrophage, wild-type recipients which limits subsequent inflammatory tissue damage. However, were reconstituted with a 1:1 mixture of bone marrow cells from NO also inhibits inflammation by repressing the Caspase1- congenically marked wild-type or mutant mice lacking either dependent processing of pro-interleukin (IL)-1β, and this Nos2 or the IFN-γ receptor (Ifngr). After a month of infection, activity prevents persistent neutrophil recruitment2. We sought to chimaerism of the CD11b+ myeloid compartment was maintained, quantify the individual contribution of these two distinct activities indicating that both genotypes populated the lung and that no obvious of NO. differences in cell death were apparent (Supplementary Fig. 2b,c). At 1Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. 2Guangdong Key Lab of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen 518112, China. 3Public Health Research Institute Center at the International Center for Public Health, New Jersey Medical School – Rutgers, New Jersey 07103, USA. 4Proteomics and Mass Spectrometry Facility, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 5Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. 6Department of Pathogen Biology, Shenzhen University School of Medicine, Shenzhen 518060, China. *e-mail: [email protected]; [email protected] NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 1 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE MICROBIOLOGY a b * WT Nos2 KO * P = 0.9773 * 105 105 P = 0.9903 * 5.63 18.2 * * P = 0.6837 ) 104 104 40 0 ) 9 P = 0.9999 P = 0.9811 10 7 * 10 * 3 3 Ly-6G 10 10 ) 6 102 102 30 P = 0.9348 0 0 −10 8 P = 0.7896 P = 0.3699 0 103 104 105 0103 104 105 20 CD11b 6 + Weight loss (%) (Gated on CD11b ) C.f.u./lungs (log −20 7 C.f.u./spleen (log 105 105 25.6 64.9 10 104 104 PMNs/lungs (×10 103 103 Ly-6G 0 −30 6 5 102 102 0 0 WT KO KO WT WT KO KO WT WT KO KO WT → → → → → → → → → → → → 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 KO WT>KOKO>KOKO>WT KO KO WT WT KO WT>WT WT WT KO WT WT KO Ly-6C cde ) 1:1 mix of BM cells ** ** ) ** 10 1 10 3 WT (CD45.1) 10 KO (CD45.2) 0 Wild type (WT) Knockout (KO) 2 (CD45.1) (CD45.2) P = 0.9903 −10 3 ** ** 0 1 Reconstitute c.f.u. −20 with mixed BM 10 Weight loss (%) 0 Wild type −30 (CD45.1) Mtb infection 2 Fold c.f.u. relative to WT (log Fold PMNs relative to WT (log Sort WT and KO −1 −40 −1 cells and determine c.f.u. Normalized log Knockout cells Knockout (CD45.2) in each population (per 1,000 sorted cells) WT WT WT Wild-type cells (CD45.1) Untreated Untreated Untreated 1 GemcitabineGem+IL-1Rn GemcitabineGem+IL-1Rn GemcitabineGem+IL-1Rn f Total lung leukocytes WT Ifngr Nos2 Nos2 KO Nos2 KO Nos2 KO 100 Unstained g hi Isotype 80 ** ** WT 25 * * ** 60 Nos2 KO 8 8 P = 0.7029 7 ) ) ) ) 40 6 10 10 20 10 20 Percentage of max 7 0 15 0102 103 104 105 7 6 CXCR2 10 5 6 C.f.u./lungs (log C.f.u./lungs (log C.f.u./lungs (log 10 PMNs/lungs (×10 104 5 103 0 6 5 5 102 101 100 Isotype Isotype Isotype CXCR2 MFI (PMNs) Untreated Untreated SCH527123 SCH527123 Anti-Ly-6G Anti-Ly-6G Anti-Ly-6G WT KO Nos2 KO Nos2 KO WT Nos2 KO C3HeB/FeJ Nos2 Figure 1 | Anti-inflammatory activity of Nos2 protects mice from TB disease. a, CD11b+ and Ly6-Ghi (insets in top panels), Ly-6C+ and Ly-6G+ (insets in bottom panels) neutrophils accumulate in the lungs of Mtb-infected Nos2−/− mice (gated on singlet/live cells). b, Bone marrow chimaeric mice were infected with Mtb (notation indicates bone marrow donor genotype → recipient genotype) and lung neutrophils, percentage weight loss and total bacterial burden in the lungs and spleen (expressed in colony-forming units, c.f.u.) were assessed 4 weeks after infection. Values are presented as mean ± s.d. *P <0.05,**P < 0.01, one-way ANOVA with Tukey’s multiple comparison test. c, Schematic for generation of mixed bone marrow chimaeric mice. d, C.f.u. levels were determined in purified haematopoietic cells of indicated genotypes. **P < 0.01, two-way ANOVA with Bonferroni multiple comparison test. e, Mtb infected Nos2−/− mice were treated with gemcitabine (gem), either alone or in combination with IL-1Rn. After 4 weeks of infection, the indicated metrics of disease were quantified. Values (mean ± s.d.) are pooled from two independent experiments. **P < 0.01, one-way ANOVA with Tukey’s multiple comparison test. f, CXCR2 surface expression was determined in CD45+ lung leukocytes (top panel) and mean fluorescence intensity (MFI) of CXCR2 in CD45+ CD11b+ Ly-6G+ F4-80− neutrophils (bottom panel) obtained at 4 weeks post infection. Data are shown as mean ± s.d. g, CXCR1/2 signalling was blocked in infected Nos2−/− mice with SCH-527123 and the number of neutrophils and c.f.u. in the lung were determined after 4 weeks of infection. Values are presented as mean ± s.d. *P < 0.05, two-tailed unpaired t-test with Welch’s correction. h, Bacterial load in the lungs (mean ± s.d.) of anti-Ly-6G- or isotype-treated animals were determined by c.f.u. assay after 14 days of neutrophil depletion. All mice were infected via the aerosol route. **P < 0.001, one-way ANOVA with Tukey’s multiple comparison test. i, Bacterial burden (mean ± s.d.) in the lungs of C3HeB/FeJ mice was determined in the lungs of anti-Ly-6G- or isotype-treated animals after 14 days of neutrophil depletion.
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